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A multiplex lamp primer combination for detecting multiple influenza viruses and its application

An influenza virus and primer combination technology, applied in the biological field, can solve problems such as false positives of primers, and achieve the effects of short reaction time, simple experimental equipment, and simple result judgment

Active Publication Date: 2022-07-12
厦门健康工程与创新研究院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The existing multiple LAMP detection of influenza virus has technical problems that false positives are easily caused between primers, and the reaction speed and sensitivity need to be further improved

Method used

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  • A multiplex lamp primer combination for detecting multiple influenza viruses and its application
  • A multiplex lamp primer combination for detecting multiple influenza viruses and its application
  • A multiplex lamp primer combination for detecting multiple influenza viruses and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] This example provides a multiplex LAMP primer combination for detecting multiple influenza viruses, refer to figure 1 As shown, in this example, a pair of primers F4 and B4 are added to the periphery of the traditional F3 and B3 to improve the reaction speed and sensitivity. The difficulty of adding a pair of primers mainly lies in primer design. Too many primers can easily cause primer dimers. The primer sequences of the final design and screening of the multiple LAMP primer combinations obtained in this example are shown in Table 1.

[0058] Table 1 Primer sequences of the multiplex LAMP primer combination of Example 1

[0059]

[0060]

[0061] Note: M1, M2, M3 are prefixes added to facilitate the distinction between the first primer set, the second primer set and the third primer set, and do not change the type of primers.

[0062] The primers of each primer set in Table 1 were artificially synthesized and used for later use. In the first primer set, the mo...

Embodiment 2

[0065] This embodiment provides a multiplex LAMP detection method, which utilizes the primer combination of Example 1 for LAMP detection, or first designs and synthesizes the multiplex LAMP primer combination of Example 1.

[0066] After obtaining the multiple LAMP primer combination of Example 1, the multiple LAMP detection method for multiple influenza virus detection specifically includes the following steps:

[0067] The multiple LAMP primer combination was formulated into primer MIX, in the primer MIX, the concentrations of FIP and BIP were 1.6 μM, the concentrations of B3 and F3 were 0.2 μM, the concentrations of LF and LB were 0.4 μM, and the concentrations of F4 and B4 were 0.3 μM; this μM is the abbreviation of μmol / L commonly used in the industry.

[0068] S2. Prepare a reaction system for loop-mediated isothermal amplification reaction. In this embodiment, the detection of the sample to be tested is taken as a test group, and the reaction system includes the DNA tem...

Embodiment 3

[0081] This embodiment provides a multiple LAMP detection method, which adds a sample collection and mixed detection step on the basis of the steps of Embodiment 2. The step is specifically: collecting samples to be tested, and mixing a plurality of samples to be tested for mixed testing. The number of samples to be tested is 5 or 10.

[0082] Mixed detection is a common method used to reduce the number of detections, and is often used in conjunction with PCR (Polymerase ChainReaction, polymerase chain reaction) detection. However, the disadvantage of mixed detection is: mixing the samples will reduce the concentration of the sample to be tested, which may be lower than the lower limit of PCR detection; resulting in an unsatisfactory mixed detection effect; but the sensitivity (lower detection limit) of LAMP is 100 times higher than that of conventional PCR, so the mixed detection is suitable for The effect of LAMP test results is much smaller than that of PCR test.

[0083]...

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Abstract

The invention discloses a combination of multiple LAMP primers for detecting multiple influenza viruses and its application. The traditional 2 pairs of primers in 6 regions are improved into 4 pairs of primers in 8 regions, F4 and B4 primers are added, and the detection time is shortened , which improves the sensitivity of detection and does not cause false positives between primers. The multiple LAMP primer combination for detecting multiple influenza viruses provided by the present invention can detect whether it is infected with Influenza A H1N1, Influenza A H3N2 or Influenza B, which is efficient and fast, and is conducive to popularization.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a combination of multiple LAMP primers for detecting multiple influenza viruses and applications thereof. Background technique [0002] Influenza virus is a representative species of Orthomyxoviridae, referred to as influenza virus. There are three types of influenza viruses, type A and type C, among which type A is the most likely to cause epidemics, followed by type B, and type C rarely causes epidemics. [0003] Loop-mediated isothermal amplification (1oop-mediated isothermal amplification) is a simple, rapid, specific and low-cost nucleic acid amplification technology, which can be completed in the next step under isothermal conditions. The method was established by Notomi et al. in 2000. , has been widely used in pathogenic microorganisms, embryo sex identification, transgenic related detection and so on. [0004] In traditional LAMP, 4 specific primers are used to...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/701C12Q1/6844C12Q2531/119C12Q2537/143Y02A50/30
Inventor 蔡文凯雷洋陈凯君白佳委陈伟川曾晓昕石岩汪大明
Owner 厦门健康工程与创新研究院
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